Pro-ferroptotic signaling promotes arterial aging via vascular smooth muscle cell senescence

Senescence of vascular smooth muscle cells (VSMCs) contributes to aging-related cardiovascular diseases by promoting arterial remodelling and stiffness. Ferroptosis is a novel type of regulated cell death associated with lipid oxidation. Here, we show that pro-ferroptosis signaling drives VSMCs senescence to accelerate vascular NAD+ loss, remodelling and aging. Pro-ferroptotic signaling is triggered in senescent VSMCs and arteries of aged mice. Furthermore, the activation of pro-ferroptotic signaling in VSMCs not only induces NAD+ loss and senescence but also promotes the release of a pro-senescent secretome. Pharmacological or genetic inhibition of pro-ferroptosis signaling, ameliorates VSMCs senescence, reduces vascular stiffness and retards the progression of abdominal aortic aneurysm in mice. Mechanistically, we revealed that inhibition of pro-ferroptotic signaling facilitates the nuclear-cytoplasmic shuttling of proliferator-activated receptor-γ and, thereby impeding nuclear receptor coactivator 4-ferrtin complex-centric ferritinophagy. Finally, the activated pro-ferroptotic signaling correlates with arterial stiffness in a human proof-of-concept study. These findings have significant implications for future therapeutic strategies aiming to eliminate vascular ferroptosis in senescence- or aging-associated cardiovascular diseases.

(C) Intracellular levels of NAD + in mouse VSMCs treated with PBS or Ang II+Bleo for 5 days.(D) Cell proliferation was measured by evaluating EdU incorporation.Mouse VSMCs treated with PBS or Ang II+Bleo for 5 days and labeled with EdU (Click-iT EdU, Invitrogen) for 1 h and processed to detect incorporated EdU.Images were obtained by fluorescent microscopy, and the number of EdU-positive nuclei was counted.n = 6 biologically independent samples.(E) Cell viability was measured using CCK-8 assay.Mouse VSMCs treated with PBS or Ang II+Bleo for 5 days and cultured in CCK-8-added medium for 1 hour.Th absorption at 450 nm was recorded using microplate reader.n = 6 biologically independent samples.(F) FerroOrange probe showing the intracellular ferrous iron pool in mouse VSMCs treated with PBS or Ang II+Bleo for 5 days.n = 5 biologically independent samples.Scale bar, 100 µm.(G) Intracellular GSH/GSSG ratio in mouse VSMCs treated with PBS or Ang II+Bleo for 5 days.n = 8 biologically independent samples.(H) Immunoblotting analyses of pro-ferroptosis factors ACSL4, ALOX15, TFR1 and antiferroptosis factor GPX4 in mouse VSMCs treated with PBS or Ang II+Bleo for 5 days.Tubulin was used as a loading control.n = 3 biologically independent samples.Data expressed the mean±SEM.*P<0.05,**P<0.01.Comparisons of parameters were performed with Two-sided Unpaired t-test.Ang II, angiotensin II; Bleo, bleomycin; GPX4, glutathione peroxidase 4; ACSL4, acyl-CoA synthetase long-chain family member 4; ALOX15, arachidonate 15-lipoxygenase; TFR1, transferrin receptor; MDA, malondialdehyde.Doses used for cell culture: Ang II, 0.1 µM; Bleo, 100 nM.

Supplemental Figure 8 Supplemental Figure 8. VSMC-specific overexpression of GPX4, but not catalytically inactive mutant GPX4Cys, inhibits vascular ferroptosis stress.
(A) Study design of AAV treatment in mice with experimental vascular senescence.The AAV particles (3 × 10 11 vg per mouse, for twice) were injected from tail vein and three days later the mice were infused with Ang II+Bleo to induce vascular senescence.At 20 days later, the mice were sacrificed for examination.(B) Confirmation of VSMC-specific overexpression of GPX4 in mice by ZsGreen1 immunofluorescent analysis.In the immunofluorescence analysis, the green signals (ZsGreen1) were noted in the tunica media of aortae.The GA The serotype 2/9 of adenoassociated virus (AAV2/9) carrying coding sequences of GPX4 wt or GPX4 cys under SMCspecific SM22a promoter (AAV-ZsGreen1-SM22α-GPX4 wt and AAV-ZsGreen1-SM22α-GPX4 Cys ) was injected.The control AAV (AAV-ZsGreen1-SM22α-CTRL) was also constructed.ZsGreen1 is a fluorescent protein used for tracing.Scale bar, 50 µm.Experiments were repeated for three times.(C) Confirmation of VSMC-specific overexpression of GPX4 in mice by immunoblotting analysis.In immunoblotting analysis, the tunica media (M) and perivascular adipose tissue (P) were isolated respectively for immunoblotting analysis.Experiments were repeated for three times.(D) Immunoblotting analyses of ferroptosis markers ACSL4 and TFR1 in aortae of mice were infused with Ang II+Bleo and with AAV injection.n = 4 biologically independent samples.(E) Relative MDA contents in aortae of mice were infused with Ang II+Bleo and with AAV injection.n = 4 biologically independent samples.(F) Study design of AAV treatment in aged mice.The AAV particles were injected for three times (every two months, 3 × 10 11 vg per mouse) in 18-month-old mice.At 6 months later (three injections), the mice were sacrificed for examination of vascular aging.Data expressed the mean±SEM.**P<0.01.Comparisons of parameters were performed with One-Way ANOVA followed by a Tukey's multiple comparisons test.
(D) Representative image and quantitative analysis of AAA diameter of enlarged aorta from WT and R26-GPX4 mice.n = 6 biologically independent samples.(E) NAD + content in AAA aortae of WT and R26-GPX4 mice.n = 6 biologically independent samples.(F) Representative images and quantitative analysis of HMGB1 immunohistochemistry staining in the AAA lesion of WT and R26-GPX4 mice.Scale bar, 100 µm.n = 6 biologically independent samples.(G) Representative images of EVG staining and quantitative analysis of elastic degradation grade in AAA tissue of WT and R26-GPX4 mice.Scale bar, 500 µm.n = 8 biologically independent samples.Data expressed the mean±SEM.**P<0.01.Comparisons of parameters were performed with One-Way ANOVA followed by a Tukey's multiple comparisons test.
senescence.ROS scavenger NAC (5 mM) or NAD + precursor NMN (300 µM) was simultaneously administrated into the medium.Five days later, the SA-β-gal staining was performed in the VSMCs.n = 8 biologically independent samples.(E-F) SA-β-gal staining of VSMCs as indicated in the figure.Ang II plus bleomycin (Ang II+Bleo) were added into the culture medium to induce senescence for 5 days.Then, ROS scavenger NAC (5 mM, E) or NAD + precursor NMN (300 µM, F) was then administrated into the medium for additional 2 days.The SA-β-gal staining was performed in the VSMCs.n = 7-8 biologically independent samples.Data expressed the mean±SEM.**P<0.01.Comparisons of parameters were performed with Two-sided Unpaired t-test.NS, no significance.

Supplemental
Representative images of H & E staining in lung of the mice with Ang II+Bleo.Scale bar, 200 µm.Experiments were repeated at least for three times.(I) Representative images of H & E staining in kidney of the mice with Ang II+Bleo.Scale bar, 200 µm.Experiments were repeated at least for three times.Data expressed the mean±SEM.**P<0.01.Comparisons of parameters were performed with Two-sided Unpaired t-test.Ang II, angiotensin II; Bleo, bleomycin; GPX4, glutathione peroxidase 4; ACSL4, acyl-CoA synthetase long-chain family member 4; ALOX15, arachidonate 15-lipoxygenase; Doses used for osmotic pump infusion: Ang II, 400 ng/kg/min; Bleo, 40 ng/kg/min.Supplemental Figure 3 Supplemental Figure 3. Treatment of angiotensin II (Ang II) plus bleomycin (Bleo) promotes ferroptosis stress, senescence and NAD + loss in aged mice.(A) Fluorescent immunocytochemistry and quantitative analysis of ferroptosis marker ACSL4 in aortae of mouse infused with saline (Control) or Ang II+Bleo for 2 weeks.Nuclei were stained by DAPI.Scale bar, 100 µm.n = 6 biologically independent samples.(B) GSH contents in aortae of mouse infused with saline (Control) or Ang II+Bleo for 2 weeks.n = 6 biologically independent samples.(C) Fluorescent immunocytochemistry and quantitative analysis of senescence marker H2A.X in aortae of mouse infused with saline (Control) or Ang II+Bleo for 2 weeks.Nuclei were stained by DAPI.H2A.X colocalizes with DAPI (nuclei).Scale bar, 100 µm.n = 6 biologically independent samples.(D) Levels NAD + in aortae of mouse infused with saline (Control) or Ang II+Bleo for 2 weeks.n = 6 biologically independent samples.(E) Immunoblotting analysis of two NAD + consumers CD38 and PARP-1 in aortae of mouse infused with saline (Control) or Ang II+Bleo for 2 weeks.n = 6 biologically independent samples.Data expressed the mean±SEM.**P<0.01.Comparisons of parameters were performed with Two-sided Unpaired t-test.Supplemental Figure 4 Supplemental Figure 4. Ferroptosis inhibitor liproxstain-1 attenuates oxidative stress, NAD + loss and senescence in cultured VSMCs.(A) Representative SA-β−gal staining and quantitative analysis in VSMCs.Scale bar, 200 µm.n = 8 biologically independent samples.(B) Representative fluorescent immunohistochemistry images and quantitative analysis of γH2A.X in mouse VSMCs.VSMCs were stained by primary antibody against γH2A.X followed with Alexa Fluor 555-conjugated second antibody (Red).DAPI was used to stain nuclei (Blue).The nuclear staining of γH2A.X colocalized with DAPI.Scale bar, 100 µm.n = 10 biologically independent samples.(C) Cell proliferation was measured by evaluating EdU incorporation assay.n = 5 biologically independent samples.(D) Cell viability was measured by evaluating CCK-8 assay.(E) Immunoblotting analysis of senescence molecular markers p53, p16 INK4A and p21 WAF1 in VSMCs.Experiments were repeated for three times.(F) Immunoblotting analysis of released SASP factors (IL-1α and HMGB1) into culture medium.Experiments were repeated for three times.

Figure 6 Supplemental Figure 6 .
Knockin of GPX4 ameliorates ferroptosis stress in mice with experimental vascular senescence via infusion of Ang II + bleomycin and natural aged mice.(A) Immunoblotting analysis of pro-ferroptosis factors ACSL4, ALOX15, 4-HNE and TFR1 in aortae from WT and GPX4 knock-in mice infused with saline or Ang II+Bleo.n = 3 biologically independent samples.(B) Intracellular GSH/GSSG ratio in aortae from WT and GPX4 knock-in mice infused with saline or Ang II+Bleo.n = 6 biologically independent samples.(C) Immunoblotting analysis of ferroptosis markers GPX4, ACSL4, ALOX15 and TFR1 in aortae from aged WT and GPX4 knock-in mice.n = 3 biologically independent samples.(D) Intracellular GSH/GSSG ratio in aortae from aged WT and GPX4 knock-in mice.n = 6 biologically independent samples.Influence of overexpression of GPX4 on SA-β-gal activity in VSMCs.n = 8 biologically independent samples.(B) Immunoblotting analysis of senescence markers p53, p21 WAF1 and p16 INK4A in VSMCs.n = 3 biologically independent samples.(C) Representative immunofluorescent images and quantitative analysis showing the influence of GPX4 overexpression (OE) on γH2A.X in VSMCs.Scale bar, 100 µm.n = 10 biologically independent samples.(D) Cell proliferation was measured by evaluating EdU incorporation assay in VSMCs.n = 4 biologically independent samples.(E) Cell viability was measured by evaluating CCK-8 assay in VSMCs.n = 5 biologically independent samples.
Figure 13 Supplemental Figure 13.Inhibition of ferroptosis signaling regulates NCOA4 expression.(A) Immunoblotting analysis of NCOA4 in cultured VSMCs with Ang II+Bleo stress, overexpression of PPARγ and PPARγ selective agonist rosiglitazone (ROSI, 10 mM).(B) Immunoblotting analysis of NCOA4 in in cultured VSMCs treated with Ang II+Bleo or overexpression of GPX4.(C) Immunoblotting analysis of NCOA4 in cultured VSMCs with Ang II+Bleo stress, overexpression of GPX4, PPARγ selective antagonist T0070907 (10 mM) and knockdown of PPARγ with siRNA.(D) Effects of ferroptosis inducer RSL3, Ang II+Bleo, GPX4 overexpression, PPARγ selective agonist rosiglitazone (ROSI) and overexpression of PPARγ on senescence markers p16 INK4A and p21 WAF1 in cultured VSMCs.All experiments were repeated for three times.Supplemental Figure 14 Supplemental Figure 14.Analysis of altered genes in aorta of mice treated with a PPARγ agonist rosiglitazone or vehicle based on a public gene expression data set (GSE1011).(A) Volcano plot shows the upregulated and downregulated genes by rosiglitazone treatment for 21 days in mice aorta./www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE1011) showing the upregulated or downregulated genes by rosiglitazone (ROSI) treatment in mice aorta.(C) Correlation plot shows the correlation between the upregulated and downregulated genes by rosiglitazone treatment in mice aorta.Supplemental Figure 15 Supplemental Figure 15.Confirmation of anti-NCOA4 in VSMCs with NCOA4 knockout by CRISPR/Cas9.For depleting NCOA4 in cells, the mouse VSMCs was transfected with CRISPR/Cas9 plasmid (Addgene) targeting NCOA4.Two days later, cells were treated with puromycin (2 µg/ml) for 5 days to select positive clones.The survived cells (NCOA4-null cells)were kept for culture and expanded.The cells were stained with anti-NCOA4 (#sc-373739, Santa Cruz Biotechnology) followed by Alexa Fluor 555-conjugated secondary antibody.DAPI was used to stain nuclei.Images were obtained in FluoView™ FV1000 Confocal (Olympus, Tokyo, Japan).Experiments were repeated for three times.